Biochemical and Biophysical Research Communications xxx (2016) 1e9

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Biochemical and Biophysical Research Communications

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1, 25(OH)2D3-induced interaction of vitamin D with p50 subunit of NF-kB suppresses the interaction between KLF5 and p50, contributing to inhibition of LPS-induced macrophage proliferation

Dong Ma a, b, Ruo-nan Zhang a,YaWenc, Wei-na Yin d, Disi Bai b, Guo-ying Zheng b, * Jin-shui Li a, Bin Zheng a, Jin-kun Wen a, a Department of Biochemistry and Molecular Biology, Key Laboratory of Neural and Vascular Biology, Ministry of Education, Hebei Medical University, Shijiazhuang, 050017, PR China b School of Public Health, North China University of Science and Technology, Tangshan, Hebei, 063000, PR China c Department of Neurology, Second Hospital of Hebei Medical University, Shijiazhuang, 050017, PR China d Department of Pediatrics, Handan First Hospital, 056000, PR China article info abstract

Article history: KLF5 and nuclear factor kB (NF-kB) regulate cell proliferation and inflammation. Vitamin D signaling Received 7 November 2016 through (VDR) exerts anti-proliferative and anti-inflammatory actions. However, an Accepted 12 November 2016 actual relationship between KLF5, NF-kB and VDR in the inflammation and proliferation of macrophages Available online xxx is still unclear. Here, we showed that LPS and proinflammatory cytokines stimulate KLF5 expression in macrophages, and that 1, 25(OH)2D3 suppresses LPS-induced KLF5 expression and cell proliferation via Keywords: upregulation of VDR expression. Mechanistic studies suggested that KLF5 interacts with p50 subunit of 1,25(OH) D 2 3 NF-kB to cooperatively induce the expressions of positive cell cycle regulators cyclin B1 and Cdk1/Cdc2 in Vitamin D receptor NF-kB LPS-treated macrophages. Further studies revealed that 1, 25(OH)2D3-induced interaction of VDR with KLF5 p50 decreases LPS-induced interaction of KLF5 with p50. Collectively, we identify a novel regulatory Macrophage pathway in which 1, 25(OH)2D3 induces VDR expression and promotes VDR interaction with p50 subunit Proliferation of NF-kB, which in turn attenuates the association of KLF5 with p50 subunit of NF-kB and thus exerts anti-inflammatory and anti-proliferative effects on macrophages. © 2016 Elsevier Inc. All rights reserved.

1. Introduction kB (NF-kB) family of transcription factors, particularly p65 and p50, are well known to regulate cell proliferation, inflammation, im- Inflammation is a fundamental adaptation to the loss of cellular mune responses, and tumor development [4,5]. For example, recent and tissue homeostasis with many important physiological roles, evidence shows that collecting duct expression of Klf5 is essential including host defence, tissue remodeling and repair, and the for inflammatory responses to the unilateral ureteral obstruction regulation of metabolism [1]. Macrophages are crucial mediators of (UUO) [6]. NF-kB plays a central role in upregulating the expression the inflammatory response and play an important role in a broad of adhesion molecules and other pro-inflammatory [7]. spectrum of acute (e.g., pathogen infection, sepsis) and chronic However, the role of NF-kB and KLF5 in linking inflammation and inflammatory responses (e.g., insulin resistance, atherosclerosis, cell proliferation had not yet been shown. chronic wounds, and tumorigenesis) [2]. Accumulating evidence Although it has been known for many years that the biologically shows that the inflammatory response requires the activation of a active vitamin D metabolite d 1,25-dihydroxyvitamin D3 complex transcriptional program that is both cell-type- and (1,25(OH)2D3) is associated with systemic calcium homeostasis, stimulus-specific and involves the dynamic regulation of hundreds there are now increasing evidences showing that 1, 25(OH)2D3 of genes [3]. Zinc finger KLF5 and nuclear factor regulates cell proliferation, differentiation, apoptosis and immune responses [8]. Previous studies have shown that the biological ef- fects of 1,25(OH)2D3 are mediated by the vitamin D receptor (VDR), which is present in many cells, including macrophages, car- * Corresponding author. E-mail address: [email protected] (J.-k. Wen). diomyocytes, vascular smooth muscle cells, and endothelial cells http://dx.doi.org/10.1016/j.bbrc.2016.11.069 0006-291X/© 2016 Elsevier Inc. All rights reserved.

Please cite this article in press as: D. Ma, et al., 1, 25(OH)2D3-induced interaction of vitamin D receptor with p50 subunit of NF-kB suppresses the interaction between KLF5 and p50, contributing to inhibition of LPS-induced macrophage proliferation, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.069 2 D. Ma et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e9

[9], and that vitamin D signaling through VDR exerts anti- 2.5. MTS assay proliferative, anti-inflammatory, pro-apoptotic, and pro- differentiating actions [10]. Mechanistic study shows that VDR is After appropriate treatment, viability of the macrophages directly involved in the regulation of nuclear factor-kB (NF-kB) cultured in 96-well plates was measured using the MTS assay, as activation [11]. Although NF-kB and VDR are well studied with previously described [14]. In brief, the medium of cultured mac- respect to their role in macrophages, the actual relationships be- rophages was replaced with 100 mL serum-free 1640 medium tween KLF5, NF-kB, and VDR in the determination of macrophage containing 10 mL of Cell Titer 96 Aqueous one solution (Promega, fate are not yet completely elucidated. G3582) and incubated at 37 C for 4 h. Then, 60 mL of medium from In this study, we tested KLF5 expression and cell proliferation in each well was transferred to a new 96-well plate, and the absor- LPS-induced macrophages and further dissected the molecular bance at 490 nm was measured using a Multiskan spectrum mechanism underlying the regulation of KLF5 expression and cell (Thermo). proliferation by 1, 25(OH)2D3. We show for the first time that 1, 25(OH)2D3 promotes competition of VDR with KLF5 for binding to 2.6. Cell cycle analysis p50 subunit of NF-kB, contributing to inhibition of LPS-induced macrophage proliferation. Cells were treated with the indicated conditions and harvested by trypsinization, and then cells were washed with ice-cold PBS, fixed with ethanol, incubated for 5 min with 0.5% Triton X-100 and 2. Materials and methods stained with propidium iodide in PBS containing 25 mg/mL RNase. Stained cells were analyzed by flow cytometry (Becton Dickinson, 2.1. Reagents San Jose, CA, USA) [15]. Ultrapure E. coli LPS was purchased from Invitrogen (San Diego, 2.7. RNA isolation and RT-PCR USA), and 1,25(OH)2D3 (VitD), cycloheximide (CHX), actinomycin D (ActD) and DAPI were purchased from Sigma (St. Louis, MO). RPMI Total RNA was extracted from macrophages using the TRizol 1640 medium and fetal bovine serum (FBS) were obtained from according to the manufacturer's instructions. 1 mgofRNAwas Hyclone Laboratories, Inc. TRIzol RNA extraction reagent was ob- subjected to reverse transcription using first-strand cDNA synthesis tained from Molecular Research Center, Inc. The reverse tran- kit (Invitrogen) according to the manufacturer's instructions. qRT- scription system was obtained from Promega. PCR of mRNAs was performed using Platinum SYBR Green qPCR Super Mix UDG Kit (Invitrogen), and real-time PCR experiments 2.2. Cell culture and treatment were carried on a ABI 7500 FAST system (Life Technologies). Rela- tive amount of transcripts was normalized with GAPDH and Murine macrophage cells (RAW264.7) were routinely cultured calculated using the 2 DDCt formula as previously described [16]. All in RPMI 1640 medium containing 10% FBS at 37 C in a 5% CO2 PCRs were performed in triplicate. The primer sequences were as incubator. Cells were treated for different times or concentrations follows: mouse GAPDH, 50-CGTCCCGTAGACAAAATGGT-30 (forward) with LPS (1 mg/mL) in 2% FBS-supplemented medium. Macrophages and 50-GAGGTCAATGAAGGGGTCG-30 (reverse); mouse KLF5, 50- were also stimulated with TNF-a (10 ng/mL) and IFN-g (400 IU/mL) ACCAGACGGCAGTAATGGACAC-30 (forward) and 50-ATTGTAGCGG- 0 0 in the presence or absence of 1,25(OH)2D3 (10e100 nM). CATAGGACGGAG-3 (reverse); mouse VDR, 5 -CACAAGACCTACGA- CCCCAC-30 (forward) and 50-CATCATGTCCAGTGAGGGGG-30 (reverse); mouse PCNA, 50-AGAAGAGGAGGCGGTAACCA-30 (for- 2.3. Transient transfection of macrophages ward) and 50- GGAGACAGTGGAGTGGCTTT-30 (reverse). Ad-KLF5 and Ad-GFP were made as described previously [12]. 2.8. Western blot analysis Macrophages were infected with Ad-KLF5 or Ad-GFP as described previously [13]. Small interfering RNAs (siRNAs) specific for mouse The cell lysates were separated by SDS-PAGE, and transferred VDR and KLF5 were synthesized by Gene Pharma (Shanghai, China). onto PVDF membrane (Millipore). Membranes were blocked with Non-specific siRNA (NS siRNA) was purchased from Santa Cruz 5% milk in TTBS for 2 h at 37 C and incubated overnight at 4 C with Biotechnology. The siRNA sequences used in these studies were as the following primary antibodies: rabbit anti-KLF5 (1:1000, follows: VDR siRNA: 50-CCCUUCAAUGGAGAUUGCCGCAUCA-30 and GTX103289, GeneTex), mouse anti-VDR (1:1000, ab-89626, 50-UGAUGCGGCAAUCUCCAUUGAAGGG-30; KLF5 siRNA: 50- GUUC- Abcam), rabbit anti-p50 (1:500, ab-38338, Abcam), mouse anti- CACAGACGUCAAUGATT-30 and 50-UCAUUGACGUCUGUGGAACTT- PCNA (1:1000, ab-18197, Abcam), rabbit anti-Cdc2 (1:1000, 30; NS-siRNA: 50-UUCUCCGAACGUGUCACGUTT-30 and 50-ACGU- 10762-1-AP, Proteintech), rabbit anti-cyclin B1 (1:1000, sc-70898, GACACGUUCGGAGAATT-30. Transfection was performed using Lip- Santa Cruz), rabbit anti-TLR4 (1:1000, 19811-1-AP, Proteintech), ofectamine™ reagent (Invitrogen) according to the manufacturer's rabbit anti-p-STAT5 (1:1000, sc-11761, Santa Cruz), rabbit anti- instructions. Twenty-hours after transfection, macrophages were STAT5 (1:1000, 12071-1-AP, Proteintech) and rabbit anti-b-actin treated with LPS (1 mg/mL). Then cells were harvested and lysed for (1:5,000, Proteintech). Membranes were washed and incubated Western blotting or co-immunoprecipitation assays. with the appropriate horseradish peroxidase-conjugated second- ary antibody (Cell Signaling Technology). bands were 2.4. Cell counting detected by ECL plus (Thermo Scientific) and b-actin was served as an internal control for protein loading. The cell number was determined using a Countess™ automated cell counter (Invitrogen) after treatment with LPS, 1,25(OH)2D3 or 2.9. Co-immunoprecipitation assay LPS plus 1,25(OH)2D3 for the different periods. Untreated cells were used to obtain baseline count. Each sample was counted three Co-immunoprecipitation was performed as described previ- times, and the average value from triplicate experiments was ously [17]. The cell lysates were immunoprecipitated with anti-p50 measured. antibodies for 1 h at 4 C, followed by incubation with protein

Please cite this article in press as: D. Ma, et al., 1, 25(OH)2D3-induced interaction of vitamin D receptor with p50 subunit of NF-kB suppresses the interaction between KLF5 and p50, contributing to inhibition of LPS-induced macrophage proliferation, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.069 Download English Version: https://daneshyari.com/en/article/5505829

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